Focal adhesions are central elements of the cell adhesion process, functioning as structural links between the cytoskeleton and extracellular matrix and triggering essential signaling pathways. Because the biophysical and biochemical processes in the focal adhesion complex are tightly coupled, mechanical analyses of adhesion provide critical information on structure-function relationships for these specialzed structures. The objective of this application is to analyze the role of focal adhesion size, position, and composition in adhesion strengthening. Our central hypothesis is that focal adhesion size, position, and composition regulate adhesion strength by controlling the distribution of mechanical loading. The rationale for this research is that, once a quantitative understanding of how focal adhesions regulate adhesion strength is established, it will be possible to systematically analyze the specific roles of focal adhesion components as well as identify strategies to control adhesion. Innovative bioengineering approaches (hydrodynamic adhesion assay, micropatterned surfaces) d be integrated with unique molecular/cell biology reagents to manipulate focal adhesion assembly in order to analyze the function of these structures. Aim 1: Analyze the effects of modulating focal adhesion size and position on adhesion strength. We hypothesize that focal adhesion size and position strongly regulate adhesion strength by reinforcing adhesion clusters and distributing mechanical forces. Aim 2: Elucidate the regulatory role of focal adhesion composition in adhesion strengthening. We will test the hypothesis that focal adhesion composition is critical for regulation of adhesion strength by analyzing the role of regulatory molecules (Rho-kinase, PI %kinase, FAJS) that significantly alter focal adhesion composition. We expect this research to yield two outcomes: (i) a quantitative understanding of the regulatory role of focal adhesions in adhesion strength and (ii) a rigorous meshwork for the analysis of adhesive mechanisms and functional studies of structural and signaling components in physiological and pathological processes.